The present invention relates to an immunogenic determinant. More particularly, it relates to a protein or a protein fragment for use in the diagnosis of Kaposi""s sarcoma or human herpesvirus 8 (KSH/HHV8) and to a diagnostic kit utilising the protein or protein fragments of the invention.
Kaposi""s sarcomaxe2x80x94relates herpesvirus (KSHV) or Human herpesvirus 8 (HHV 8) may be the postulated infectious cause of Kaposi""s Sarcoma and is closely related to Epstein-Bar virus (HHV 5). Its prevalence in the general population is controversial. Antibodies to latent KSHV/HHV 8 antigen(s) are largely restricted to individuals with, and at risk for, KS. The antibody response to different EBV antigens (viral capsid, early, latent antigen) varies in individuals with acute, latent, or reactivated EBV infection.
Epidemiological evidence suggest that Kaposi""s sarcoma in both HIV-uninfected and -infected individuals is caused by a transmissible agent: Among HIV-infected patients KS is much more common in gay men than in other HIV transmission groups, in particular haemophiliacs, transfusion recipients and intravenous drug users (IVDU). Transmission of the putative KS agent occurs independently of that of HIV and shows a marked geographic variation. Similarly, the incidence of KS outside HIV infection differs dramatically in different geographic areas: In Africa, it is higher in central and Eastern Africa than in West or South Africa, and in Europe, it is commoner in some Mediterranean countries.
The recently discovered Kaposi Sarcoma associated herpesvirus (KSHV), or human herpesvirus 8 (HHV8) is a xcex32-herpesvirus and closely related to EBV. KSHV/HHV 8 may be the infectious cause of KS, as it is consistently found in all forms of Kaposi""s sarcoma, i.e. AIDS-related KS, African endemic KS, classic HIV-negative KS and KS in transplant patients. KSHV/HHV 8 genomes are present in endothelial and spindle cells, the histological hallmark of KS lesions. Detection of KSHV/HHV 8 in peripheral blood correlates with, and in asymptomatic HIV-infected individuals predicts the development of, KS lesions. However, whether the distribution of KSHV/HHV 8 matches that expected for the putative KS agent, is still controversial: while several groups have not detected KSHV/HHV 8 by PCR in the peripheral blood of healthy blood donors, others have reported its presence in 9% of PBMC and lymphoid tissue from HIV-uninfected persons. Similarly, some, but not other groups have reported a high prevalence in semen samples from healthy donors. More recently, the first serological studies have been carried out using immunoblotting and immunofluorescence assays on B-cell lymphoma cell lines, either dually infected with KSHV/HHV 8 and EBV, (Moore, P. S., Gaso, S. -J., Dominguez, G., et al. Primary characterization of a herpesvirus agent associated with Kaposi""s sarcoma. J. Virol. 1996; 70: 549-558; Miller, G., Rigsby, M. O., Heston, L., et al. Antibodies to butyrate-inducible antigens of Kaposi""s sarcomaxe2x80x94associated herpesvirus in patients with HIV-1 infection. N. Engl. J. Med. 1996; 334:1292-1297; Gao, S. J., Kingsley, L., Hoover, D. R. et al. Seroconversion of antibodies to Kaposi""s sarcoma-associated herpesvirusxe2x80x94related latent nuclear antigens before the development of Kaposi""s sarcoma. N. Engl. J. Med. 1996; 335: 233-241) or only infected with KSHV/HHV 8 (Gao, S. J. Kingsley, L., Li, M. et al. Seroprevalence of KSHV antibodies among North Americans, Italians, and Ugandans with and without Kaposi""s sarcoma. Nature Medicine 1996; 2: 925-928 (Kedes, D. H., Operskalski, E., Busch, M. et al. The seroprevalence of human herpesvirus 8 (HHV 8): Distribution of infection in Kaposi""s sarcoma risk groups and evidence for sexual transmission. Nature Medicine 1996; 2: in press). Antibodies to latent KSHV/HHV 8 antigen(s) were found in the vast majority of KS patients, but not, or only rarely, in HIV-infected IVDU, haemophiliacs, or healthy blood donors. Seroconversion to latent nuclear antigens can occur years prior to KS ones: and is strongly predictive of subsequent disease (Gao, S. J., Kingsley, L., Hoover, D. R. et al. Seroconversion of antibodies to Kaposi""s sarcoma-associated herpesvirus-related latent nuclear antigens before the development of Kaposi""s sarcoma. N. Engl. J. Med. 1996; 335: 233-241; Gao, S. J., Kinglsey, L., Li, M. et al. Seroprevalence of KSHV antibodies among North Americans, Italians, and Ugandans with and without Kaposi""s sarcoma. Nature Medicine 1996; 2: 925-928). The antibody response to different antigen complexes (latent, capsid, early antigen) of the closely related EBV varies in acutely vs. chroncially infected individuals or immunosuppressed patients with reactivated EBV infection (Crawford, D. H. Epstein-Barr Virus in: Clinical Virology, Zuckerman, A. J., Banarvala, J. E., Pattison, J.R. eds., John Wiley and Sons, New York 1995; pp. 109-14; Horneff, M. W. Bein, G., Fricke, L., et al. Coincidence of Epstein-Barr virus reactivation, cytomegalovirus infection, and rejection episodes in renal transplant recipients. Transplantation 1995; 60: 474-480). Several potentially immunoreactive lytic-cycle proteins of KSHV/HHV 8, including the major capsid protein (MCP), are highly homologous to their EBV counterparts (Moore, P. S., Gao, S. -J., Dominguez, G., et al. Primary characterization of a herpesvirus agent associated with Kaposi""s sarcoma J. Virol. 1996; 70: 549-558) and are thus unlikely to provide specific serological antigens.
In seeking an immunogenic determinant, recombinant proteins were generated in E. Coli from a number of orf encoded genes of KSHV/HHV8.
Particularly favourable results were obtained with a recombinant protein of orf 65xe2x80x94a lytic cycle/capsid related structural protein. This was particularly surprising given that KSHV/HHV8 homologues of at least two immunoreactive EBV proteins orf 52 and orf 29b had failed to react (as recombinant proteins) with sera from patients with Kaposi""s Sarcoma.
More particularly the applicant has determined that the immunogenic region of the orf 65 protein was to be found at or within the carboxyterminal endxe2x80x94ie about the last 80 amino acids.
The amino acid sequence of the carboxyterminal end of KSHV orf 65 is given below:
Sequence 1: Amino acids 86-170 of KSHV orf 65
It is an aim of the present invention to identify proteins and/or protein fragments for use in the diagnosis of Kaposi""s Sarcoma and associated herpesvirus (KSHV/HHV8).
It is a further aim to produce diagnostic kits, such as for example, an ELISA kit or competitive assay kit using these proteins and/or protein fragments.
It is a further aim to produce monoclonal or polyclonal antibodies to these proteins and/or protein fragments.
According to a first aspect of the present invention there is provided an immunogenic determinant comprising, consisting of or containing an amino acid sequence substantially homologous with the carboxyterminal end of an orf 65 protein.
According to a second aspect of the present invention there is provided an immunogenic determinant comprising, consisting of or containing an amino acid sequence:
[SEQ ID NO: 1]
ADRVSAASAY DAGTFTVPSR PGPASGTTFG GQDSLGVSGS SITTLSSGPH SLSPASDILT TLSSTTETAA PAVADARKPP SGGKKK
According to a third aspect of the present invention there is a method of screening for an infection with KSHV and/or HHV8 which method utilises an immunogenic determinant of the invention.
According to a fourth aspect of the present invention there is provided an immunogenic determinant derived from the carboxyterminal end of an orf 65 protein for use in a vaccine or as a diagnostic tool against KSHV or HHV8.
According to a fifth aspect of the present invention there is provided an immuno-assay kit comprising an immunogenic determinant of the invention.
Preferably the kit is in the form of an ELISA or competitive assay kit.
According to a further aspect of the present invention there is provided an antibody directed to an immunogenic determinant of the invention.
Preferably the antibody is a monoclonal antibody.